Leukotrienes are metabolites of arachidonic acid formed by the enzyme 5-lipoxygenase (5-LO). Interest in these molecules continues to expand because of their biological activities as a neutrophil chemotactic factor (leukotriene B4, LTB4) and bronchial smooth muscle contraction (leukotriene C4, LTC4). The biosynthesis of leukotrienes is now known to take place on the perinuclear membrane where 5-LO, 5-LO activating protein and cytosolic phospholipase A2 are assembled to convert arachidonic acid into leukotriene A4 (LTA4). LTA4 is a conjugated triene epoxide that has considerable chemical reactivity exemplified by a chemical half-life of less than one second in aqueous buffer. Nevertheless, LTA4 can be efficiently transported from one cell to another in a process termed transcellular biosynthesis. An understanding of the biochemical events involved in transport are completely unknown, but must involve some sequestration of LTA4 from the aqueous milieu to prevent its nonenzymatic hydrolysis. A major aim is to identify and characterize proteins that stabilize LTA4 within the cell and thereby facilitate transcellular biosynthesis of leukotrienes. Mass spectrometry based proteomics will be used to identify the proteins using a LTA, half-life assay to guide purification of cytosolic proteins. The electrophilic nature of LTA4 also renders this intermediate a likely candidate to participate in covalent binding reactions with cellular macromolecules. A second specific aim is to study the extent of covalent binding of LTA4 to DNA and RNA using highly sensitive and quantitative mass spectrometric assays (LC/MSIMS) and assess production of these adducts from exogenous and endogenous LTA4. While advancements have been made in the identification of potential pathways of LTB4 metabolism in vivo, clear understanding of those LTB4 metabolites which appear in urine and could serve as markers of endogenous production of LTB4 is lacking. A new strategy will be implemented to facilitate structural identification of specific urinary metabolites. The development of a quantitative assay for such a metabolite will have a major impact on the assessment of LTB4 production in human health as well as disease.